Encapsulation of technetium-99m sestamibi (Tc-99m-MIBI) in polyethyleneglycol-liposomes (Tc-99m-MIBI-PEG-liposomes) could extend the duration of its circulation in blood and alter its biodistribution, enabling its concentration in tumours to be increased. An original method to encapsulate Tc-99m-MIBI in PEG-liposomes is described. The Tc-99m-MIBI-PEG-liposomes were compared with free Tc-99m-MIBI with respect to (a) tumour availability (b) ability to distinguish between chemotherapy-sensitive and -resistant cells and (c) uptake ratio in tumour imaging. PEG-liposomal systems composed of distearoylphosphatidylcholine/cholesterol/PEG(2000)-distearoyl phosphatidylethanolamine and lissamine-rhodamine B-labelled liposomes were used. The encapsulation of 99mTc-MIBI in liposomes was achieved using the K+ diffusion potential method. We compared the uptake of free versus encapsulated Tc-99m-MIBI by sensitive and resistant erythroleukaemia (K562) and breast tumour (MCF-7ras) cells. To assess the internalisation of these liposomes into cells, rhodamine B-labelled PEG-liposomes were used and visualised by fluorescence microscopy. Biodistribution and imaging characteristics of encapsulated and free radiotracer were determined in rats and tumour-bearing nude mice. The efficiency of Tc-99m-MIBI encapsulation in PEG-liposomes was 50+/-5%. Use of Tc-99m-MIBI-PEG-liposomes did not impair the ability of this tracer to distinguish between chemotherapy-sensitive and -resistant tumour cells; the percentage of radio-activity accumulated in the sensitive K562 cells was 1.24+/-0.04%, as compared with 0.41+/-0.04% in the resistant K562 cells. One hour post injection in rats, PEG-liposomes showed a ten times higher activity in blood than free Tc-99m-MIBI, whereas activity of free Tc-99m-MIBI in kidneys and bladder was markedly higher than that of encapsulated Tc-99m-MIBI, indicating faster clearance of the free radiotracer. In the (MCF7-ras)-bearing nude mice, PEG-liposome uptake in tumour was two times that of free Tc-99m-MIBI. Summarising, the Tc-99m-MIBI-PEG-liposomes demonstrated a longer blood circulation time, enabled distinction between chemotherapy-sensitive and -resistant cells and improved tumour to background contrast in in vivo imaging. Tc-99m-MIBI-PEG-liposomes therefore show promising potential for tumour imaging.